Protein and peptide drugs have excellent therapeutic effects that otherwise cannot be treated by general synthetic chemical drugs, and thus take important positions in medicine and pharmacy. For example, recombinant human growth hormone (hGH) is the sole effective therapeutic agent for the treatment of growth hormone deficiency, and recombinant human erythropoietin (EPO) is used in treating anemia resulting from chronic kidney disease due to its ability to increase the level of red blood cells, and recombinant granulocyte colony stimulating hormone (G-CSF) is used as the sole drug to increase the white blood cell count in cancer patients after chemotherapy. In addition, various kinds of cytokines, hormones and peptides which are found in the body are used as the sole therapeutics for a wide spectrum of diseases for which no other alternatives are currently available.
Although they exhibit excellent therapeutic effects in vivo, these protein or peptide drugs quickly lose their therapeutic activity and thus have short half-lives in vivo because they are degraded by blood proteinases immediately after injection or they are readily removed from the body by the kidney or liver. Thus, they are disadvantageous in that they require frequent injections in order to maintain a constant blood level or titer thereof. Such frequent injection lowers the drug compliance of patients because of the fear and pain of injection or inconvenience by repeated administrations when they are used for a long period of time.
Many studies have been continuously conducted in order to increase the blood stability of protein and peptide drugs and maintain the levels of the drugs in the blood for a long period of time.
For example, sustained release dosage forms of drugs have been developed by formulating a therapeutically active protein or peptide with a bio-degradable polymer that allows proteins or peptides to be slowly released from the injection site. When the sustained release drug is subcutaneously or intramuscularly injected, the drug is slowly released to maintain the drug at a constant level for a specific period of time (M. Chasin & R. Langer, et al., Biodegradable polymer as drug delivery system, Marcel Dekker (1990); J. Heller, et al., Adv. Drug Del Rev., 10, 163 (1993)). Among the bio-degradable polymers, PLGA (poly(lactic-co-glycolic acid) has been widely used. For example, a sustained dosage form of LHRH (luteinizing hormone-releasing hormone) agonist peptide was produced, and it was found that this product releases the peptide in vivo over one or three months. The use of bio-degradable polymers has been applied to large-molecular weight proteins. For example, U.S. Pat. No. 6,500,448 discloses a pharmaceutical composition for the sustained release of human growth hormone which comprises a biocompatible polymer, and particles of metal cation-complexed human growth hormone. In another study, Korean Patent Nos. 10-0236771 and 10-0329336 described the use of hyaluronic acid for the sustained release microparticles of the protein drugs, featuring the application of recombinant human growth hormone.
Even though, for the sustained release of drugs, bio-degradable polymers have been successfully applied to low-molecular weight peptides, there are limitations concerning their application to large-molecular weight proteins. The reason for this is that proteins are easily denatured in the course of producing sustained release microparticles and the denatured amino acids lower the activity of the protein, which cause some undesired immune responses in human body. In addition, the size of microparticles for the sustained release of proteins or peptides is generally large, requiring thick syringe needles when injected into human, which create pain at injection site. Also, the microparticles have the economical disadvantage of low production yields in the production of the products for the commercial purpose.
In order to overcome the aforementioned problems, studies have been directed towards the delay of renal clearance of proteins or peptides. On the whole, proteins with a molecular weight of 60,000 daltons or less pass through the kidney without renal retention. Hence, attempts have been made to enlarge the low-molecular weight of peptide or protein therapeutics to prolong in vivo circulating time, thus reducing the frequency of injection. According to these techniques, physiologically active proteins and peptides are not provided in a sustained release form but rather in a long-acting form.
One of the most popular strategies used to reduce injection frequency is to attach a highly soluble polymer such as polyethylene glycol (hereinafter referred to as “PEG”) to the surface of pharmaceutically active proteins or peptides. PEG can be non-specifically attached to the amine group of amino acids of proteins or peptides. PEGylation can provide water solubility to hydrophobic drugs and proteins and increases the hydrodynamic size of the agent to prolong the time in circulation when it is injected to the body (Sada et al., J. Ferment Bioeng 71, 137-139, 1991).
Recently, PEGylated interferon alpha has been commercialized in order to reduce the injection intervals. In addition, Kinstler et al. demonstrated that one injection of PEGylated granulocyte colony-stimulating factor (G-CSF) per week (one chemotherapy cycle) had the same medical effect as did triweekly injections of G-CSF (Kinstler et al., Pharm Res 12, 1883-1888, 1995). PEG-GCSF was commercially available under the tradename of “Neulast.”
Since the PEGylation of a protein results from the non-specific covalent conjugation of PEG to the surface of the protein, the interaction of the protein with its receptor may be hindered at the PEGylated region, thus significantly decreasing the in vivo activity of the protein. In addition, PEGylation is somewhat cumbersome because the proteins pegylated at the physiologically active site must be removed during a purification process to leave behind the PEG-protein conjugates which have their activity decreased to the minimal degree possible. In this process, thus, the production yield of desired PEG-protein conjugates is significantly lowered, resulting in the economically unfavorable situation. In addition, as for some proteins that are unstable in aqueous solutions, attempts to conjugate with PEG has been failed.
Also, a glycoengineering technique has been used to reduce injection frequency and has now been commercialized. Elliot et al. reported the additional glycosylation of erythropoietin (EPO) by substituting amino acids at certain positions (Nat Biotechnol 21, 414-421, 2003; U.S. Pat. No. 7,217,689). The erythropoietin modified by glycoengineering technique is now commercially available under the tradename of “Aranesp” and it is known that the circulation in the blood stream, metabolism and excretion of the modified erythropoietin are retarded due to the addition of sugar chains with sialic acid at the terminus and the increased molecular weight. However, the glycoenginneeing technology to introduce additional glycosilation sites of the proteins has not been widely used, because the attachment or addition of sugar chains may cause inactivation of the physiologically active protein, and its ability to maintain in vivo stability of many proteins has not been proven. And the choice of sites of the physiologically active protein to which sugar chains can be additionally attached is very narrow. In addition, the glycoengineering technology is not easy to apply to low-molecular weight peptides.
The development of genetic engineering technology has allowed to enlarge the size of a physiologically active protein by fusion with a high-molecular weight protein (Curr Opin Drug Discov Devel 12, 284-295, 2009). For example, a physiologically active protein gene is fused to a human albumin gene and then expressed in yeast cells to produce a fusion protein (International Patent Publication Nos. WO 93/15199 and WO 93/15200). Examples of the physiologically active protein fused to albumin include granulocyte colony stimulating factor (Halpern et al., Pharm Res 19, 1720-1729, 2002), human growth hormone (Osborn et al., Eur J Pharmacol 456, 149-158, 2002), glucagon like peptide-1 (Baggio et al., Diabetes 53, 2492-2500, 2004), and interferon alpha (Osborn et al., J Pharmacol Exp Ther 303, 540-548, 2002).
In the case of recombinant fusion technology, fusion proteins with transferrin are also known. For example, U.S. Pat. No. 7,176,278 discloses a fusion molecule in which glucagon like peptide-1 is fused to native transferrin or aglycosylated transferrin and becomes increased in vivo half-life.
Meanwhile, the in vivo half-life of a protein can be extended by fusion to an immunoglobulin (Ig) Fc fragment (U.S. Pat. Nos. 5,116,964 and 5,605,690). A fusion gene of TNF-α receptor fragment and IgG1 Fc fragment was expressed in an animal cell (Chinese hamster ovary, CHO) transformed with the gene encoding the fusion protein and the fusion protein is now commercially available (tradename: Enbrel) after approval of USFDA as a therapeutic agent for rheumatoid arthritis. Further, Wang (Qinghua Wang; WO 2007/012188) extended the in vivo half-life of GLP-1 (t1/2<2 min) or exendin-4 with short half-life by fusion to an Ig Fc fragment.
Even if Ig Fc is widely used as a carrier for fusion proteins in order to increase in vivo half-life, IgG1 Fc retains its own antibody-dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Hence, when injected into the body, a fusion protein of a physiologically active protein with IgG1 Fc may cause complex immune responses. In addition, repeated administration of Fc fusion proteins for a long period of time may produce undesired antibodies. Accordingly, the use of IgG1 Fc fusion proteins has limitation in clinical application.
Korean Patent No. 10-0725315 discloses a protein complex using an immunoglobulin fragment and a method for the preparation thereof in which a physiologically active protein is fused to IgG Fc via PEG. The “protein complex” having a structure of physiologically active protein-PEG-Fc has a longer in vivo half-life than the physiologically active protein as measured by pharmacokinetic assay. However, the similar drawbacks or problems shown in Fc fusion method can be also observed in “protein complex” because a physiologically active protein and an Fc fragment are chemically bonded by PEG molecule.
Another example of the use of immunoglobulin in enhancing the in vivo stability of a peptide drug is the fusion of an entire IgG antibody molecule and a low-molecular peptide (Rader et al, Proc. Natl. Acad. Sci. U.S.A. 100, 5396-5400, 2003, Doppalapudi et al., Bioorg & Med Chem 17, 501-506, 2007). However, this technique, called “CovX-Body,” cannot be applied to large-molecular weight proteins and its use is limited due to the problems generated upon the production of the Fc fusion proteins or the PEG fusion proteins.
As described above, many attempts have been made to fuse a biopolymer to a physiologically active protein or therapeutic peptide, but can be applied only to a limited range of proteins or peptides for the following reasons: in vivo residency time is not sufficiently long enough to develop the fusion protein for medicinal use; remarkably low production yield resulting in economically unfavorable situation; undesired immune responses when used for a long time; and the undesirable residual presence of toxic chemical derivatives when used for the conjugation with proteins or peptides. There is, therefore, a need for novel fusion proteins or peptides that can extend the in vivo half-life of physiologically active proteins or peptides, with the minimal loss of in vivo activity.